Erectile dysfunction (ED) is estimated to affect more than 152 million men worldwide. Diabetes is strongly associated with ED and it increases the incidence of ED by threefold. Current therapies for ED including cyclic GMP (cGMP)-specific phosphodiesterase 5 inhibitors are less effective in diabetic patients because these patients usually have a severely impaired nitric oxide (NO)/cGMP pathway. Therefore, a better understanding of physiological and molecular mechanisms of penile erection is crucial for discovering new alternative therapeutic approaches for diabetic patients with ED. It is well-established that the RhoA/Rho-kinase mediated Ca2+ sensitizing pathway maintains cavernosal smooth muscle contraction through inhibition of myosin light chain phosphatase (MLCP), leading to the maintenance of myosin light chain (MLC) phosphorylation. In this research proposal, we will focus on the novel downstream targets of RhoA/Rho-kinase, through which the RhoA/Rho-kinase pathway exerts an anti-erectile influence interacting with other erectile regulatory pathways. We hypothesize that up-regulated RhoA/Rho-kinase signaling in diabetes impairs erectile function via inhibition of endothelial NO synthase (eNOS) function and activation of NADPH oxidase, a reactive oxygen species (ROS) generation system. We will use fatty diabetic Zucker (ZDF) rats as a Type II diabetic model, which has been shown to closely mimic human onset Type II diabetes. To support our hypothesis we will transfect the penile tissue of ZDF rats in vivo with dominant-negative RhoA or small interference RNA to inhibit the RhoA/Rho-kinase pathway, preventing or reversing the imbalance between NO and ROS, which may then improve erectile function. We will also overexpress active RhoA in lean Zucker rats and young pre-diabetic ZDF rats to determine whether ED is caused in these groups and involves dys-regulation of NO and ROS production. Understanding the molecular mechanisms of RhoA pathway regulating erectile function and the effectiveness of gene therapy of Rho-kinase will provide a new therapeutic approach for treatment of ED. This study aims to understand why ED is common in diabetic men. We try to determine the mechanisms of RhoA/Rho- kinase, the major vasoconstrictive pathway, in diabetes associated ED. Erectile dysfunction (ED) is estimated to affect more than 152 million men worldwide. Diabetes is strongly associated with ED. However, diabetic patients usually respond to current therapies poorly. This study aims to understand why ED is common in diabetic men. We try to determine the mechanisms of RhoA/Rho-kinase, the major vasoconstrictive pathway, in diabetes associated ED. Understanding the molecular mechanisms of RhoA pathway regulating erectile function will provide a new therapeutic approach for treatment of ED. [unreadable] [unreadable] [unreadable]